We have demonstrated for the first time the quantitative formation of methotrexate polyglutamates in human tumor cells. The key enzyme, dCMP deaminase, which controls de novo synthesis of dCTP, has been purified to near homogeneity by a novel affinity procedure, and its regulation by nucleotide inhibitors and activators has been thoroughly characterized. Significant enhancement of fluorouracil activation to FdUMP and enhanced incorporation into RNA have been demonstrated in the presence of the new pyrimidine antagonist PALA. A clinical protocol for treatment of malignant melanoma based on these findings has indicated a response rate (objective regressions, partial response, and complete response) of 3 of 9 patients evaluable thus far. Detailed pharmacokinetic studies of PALA have been completed using a novel competitive binding assay procedure. We have confirmed earlier studies which showed the pineal secretion, melatonin, suppresses development of breast tumors in rats exposed to the carcinogen DMBA. This suppression is correlated with suppression of prolactin secretion by the pituitary and suggests that melatonin may exert an important influence over the prolactin secretory pattern in mammals.